The present invention relates to a high throughput biological heart rate monitor that is molecularly determined.
Throughout this application, various publications are referenced to by numbers. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to those skilled therein as of the date of the invention described and claimed herein.
The pacemaker current, If, is present in both automatic (1) and non-automatic (2-6) regions of the heart. Further, the threshold voltage of activation varies widely among cardiac regions, being least negative in the sinus node (e.g. in rabbit sinus node it is xe2x88x9240 mV (7)) and most negative in the ventricle (xe2x88x92108 mV or more negative, depending on species (5,8,9). Interestingly, the current activates at less negative voltages in the newborn ventricle (approximately xe2x88x9270 mV in rat (8,10)) and the diseased adult ventricle (approximately xe2x88x9270 mV threshold in aged hypertensive rat(11), xe2x88x9255 mV in failing human ventricle (12)). The molecular and cellular bases for the regional variability of activation voltages in the normal adult heart and the regulation of ventricular activation voltage by development and disease remain to be determined, but such understanding is critical to any future therapeutic application of the expressed current in myocardium.
This invention provides for a method of assaying whether an agent affects heart rate which comprises: (a) contacting a cardiac cell of a heart with an effective amount of a compound to cause a sustainable heart rate; (b) measuring the heart rate after step (a); (c) providing the heart with an agent to be assayed for its affects on heart rate; (d) measuring the heart rate after step (c); and (e) comparing the difference between step (b) and step (d), thereby determining whether the agent affects heart rate.
This invention also provides a method of assaying whether an agent affects heart rate which comprises: (a) disaggregating cardiac moyocytes from a heart; (b) measuring the beating rate of the cardiac myocytes after step (a); (c) contacting a set of the cardiac myocytes from step (a) with an agent to be assayed for its effects on heart rate; (d) measuring the heart rate after step (c); and (e) comparing the measurements from step (b) and step (d), thereby determining whether the agent affects heart rate.
This invention further provides a method of assaying whether an agent affects the membrane potential of a cell which comprises: (a) contacting the cell with a sufficient amount of a compound capable of lessening the negativity of the membrane potential of the cell; (b) measuring the membrane potential of the cell after step (a); (c) providing the cell with the agent to be assayed for its effects on the membrane potential of a cell; (d) measuring the membrane potential of the cell after step (c); and (e) comparing the difference between the measurements from step (b) and step (d), thereby determining whether the agent affects the membrane potential of the cell.
This invention further provides a method of assaying whether an agent affects the activation of a cell which comprises: (a) contacting the cell with a sufficient amount of a compound to activate the cell; (b) measuring the voltage required to activate the cell after step (a); (c) providing the cell with an agent to be assayed for its affects on the activation of the cell; (d) measuring the voltage required to activate the cell after step (c); and (e) comparing the difference between the measurements from step (b) and step (d), thereby determining whether the agent affects the activation of the cell.
This invention further provides a method of assaying whether an agent affects the contraction of a cell which comprises: (a) contacting a cell with an effective amount of a compound to contract the cell; (b) measuring the level of contraction of the cell after step (a); (c) contacting the cell with the agent to be assayed for its affects on contraction of the cell; (d) measuring the level of contraction of the cell after step (c); and (e) comparing the difference between the measurements from step (b) and step (d), thereby determining whether the agent affects the contraction of the cell.
This invention also provides a vector which comprises a compound which encodes an ion channel gene.
This invention further provides for a chamber and system designed for use in assaying drug effects on heart rate. The chamber consists of a series of wells, each 3 mm by 3 mm in inner diameter. Cardiac myocytes disaggregated from neonatal animals are plated onto the bottom of each well and grown under standard tissue culture conditions. The chamber holds from 24-96 such wells. When drugs are to be assayed, the cells in each well are loaded with a calcium sensitive dye and the beating rate in each is monitored with a photodiode. Drug is added in graded concentrations to each well, and equilibrated and effects on rate are observed. This construct permits use of a cell based bioassay for the study of drugs or agents that may alter cardiac rate. This invention can be used in high throughput screening of drugs to evaluate/predict their effects on cardiac rate and rhythm.